A specific D-glucose-inhibitable cytochalasin B binding assay for quantitating the number of glucose transporters in subcellular membrane fractions has previously been used to demonstrate that insulin stimulates glucose transport in the isolated rat adipose cell primarily through the translocation of glucose transporters from a large intracellular pool is associated with the cell's low-density microsomal membranes. This assay has now been used to demonstrate that the intracellular pool may comprise a specialized membrane species and that intracellular glucose transporters in response to insulin. Indeed, a nonfunctional translocation of glucose transporters can be induced in the absence of insulin by TRIS. TRIS also inhibits any further translocation of glucose transporters in response to insulin, but does not prevent insulin from activating those glucose transporters already present in the plasma membrane. Incubation of cells at 16 C or less is also accompanied by a rapid translocation of glucose transporters without a corresponding increase in glucose transport. In the presence of insulin, glucose transport is fully stimulated with little further translocation of glucose transporters. When cells are exposed simultaneously to insulin and either isoproterenol or adenosine deaminase, glucose transport is decreased by 25% without a corresponding decrease in the translocation of glucose transporters. In combination, however, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport by 75% but decrease the translocation of glucose transporters by only 45%. Similar effects are observed with dibutyryl cAMP. These results suggest that insulin stimulates glucose transport through a subcellular redistribution of glucose transporters rapidly cycling in an exocytic/endocytic-like fashion. Furthermore, insulin's action appears to occur at a step wher by glucose transporters associated with the plasma membrane become functional. A cAMP-mediated counterregulation of insuli-stimulated glucose transport by catecholamines, on the other hand, appears to occur in part through regulation of the subcellular distribution of those glucose transporters present in the plasma membrane. Insulin also appears to stimulte glucose transport in isolated guinea pig and human adipose cells, and rat diaphragm through this same translocation mechanism.